The coronavirus genome is highly structured and contains numerous cis acting sites that regulate virus transcription, replication, gene expression, packaging and release. Despite this highly conserved structure, we have demonstrated that the MHV and TGEV group specific ORFs encode luxury functions that can be deleted, diminishing virus replication in vitro. Moreover, we and others have shown that coronaviruses tolerate gene order rearrangements, but as a consequence are attenuated in replication efficiency and pathogenesis. Our laboratory has developed full-length infectious cDNAs for mouse hepatitis virus (MHV), transmissible gastroenteritis virus (TGEV) and severe acute respiratory syndrome coronavirus (SARS-CoV). The overall hypothesis guiding this research is that deletion or rearrangement of selected ORFs and/or rewiring of the coronavirus regulatory networks will attenuate SARS replication in vitro and will provide a novel set of candidate vaccines that protect against wild type challenge in vivo. In aim 1, we will test the hypothesis that one or more of the SARS group specific ORFs are nonessential by sequential deletion. We will characterize recombinant virus replication efficiency and gene expression in vitro and in vivo and determine if infection with these recombinant viruses protect against subsequent wild type replication in Balb-c mice. In Aim 2, we test the hypothesis that gene order rearrangements are viable and that rescued viruses replicate less efficiently in vitro and in vivo, but protect against wild type challenge in Balb-c mice. In aim 3, we test the hypothesis that SARS coronavirus genetic regulatory networks can be remodeled. We will determine if these viruses recombine with wild type SARS and unrelated coronaviruses in vitro and in vivo and protect against in vivo challenge. The overall goal of these studies is to develop a suite of molecularly cloned SARS coronaviruses that contain stably attenuating alterations in the viral genome that not only protect against wild type challenge in vivo, but also are safe and cannot recombine with circulating coronavirus strains in nature.